There is an urgent need to understand the initiation and dissemination of HIV-1 infection in mucosal tissues, and to develop new methods for targeting early infection to prevent mucosal HIV-1 transmission. HIV-1 infection requires fusion of the viral and target cell membranes, a process mediated by the viral envelope glycoprotein (Env) and host cell receptors. The human scavenger receptor gp340 has been identified as a secreted component in human saliva that inhibits oral transmission of HIV-1 through a specific interaction with the surface gp120 subunit of an Env spike. Surprisingly the gp120 interaction with the membrane-bound gp340 in the female reproductive tract promotes HIV-1 transcytosis through barrier epithelia and thus infection of CD4+ cells. Our goal in this program is to define the molecular target and mechanism of action of soluble gp340-based HIV-1 entry inhibitors. Our discovery effort will be based on preliminary data obtained with the gp120-binding SRCR domain of gp340 that potently inhibits in vitro infection by HIV-1 with nanomolar IC50 values. We propose a comprehensive, interdisciplinary approach that combines protein chemistry, high- resolution structural determination, thermodynamic and kinetic binding analyses, protein engineering, molecular virology, and animal model efficacy studies. In this project we seek to discover novel HIV-1-specific intervention strategies to inhibit mucosal epithelium-Env binding and virus-cell fusion. The Specific Aims are: 1. To understand the structural basis of gp120 binding by gp340 to inform the design and optimization of novel SRCR domain entry inhibitors. (a) Elucidate the structural properties of the SRCR domain fragment both free and bound to the V3 region. (b) Characterize the energetics of the gp120-gp340 interaction. (c) Use mutational analysis and biophysical methods to dissect the structural determinants of the respective binding sites. (d) Define a minimal gp120-binding sequence or set of such sequences. (e) Optimize binding affinity of variants of the SRCR domain, and evaluate their anti-HIV-1 activity in a single-round infectivity assay. 2. To determine the mechanisms by which the gp120-gp340 interaction influences Env structure, function and antigenicity. (a) Determine how V3 mutations identified in Aim 1c influence the functional competence of Env. (b) Characterize the effects of the SRCR domain on the receptor-binding properties of gp120 and its reactivities to neutralizing and non-neutralizing monoclonal antibodies. (c) Determine the virucidal activity of select SRCR domain entry inhibitors against diverse primary HIV-1 strains. 3. To test the effectiveness of optimized SRCR domain entry inhibitors to protect against mucosal HIV-1 infection. (a) Characterize the specificity and potency of select SRCR entry inhibitors in an in vitro model of HIV-1 infection of human cervical and vaginal tissue. (b) Use the humanized mouse vaginal transmission model to assess the in vivo potency and breadth of activity of optimized SRCR entry inhibitors alone and in combination with the gp41 fusion inhibitor C52L and the small-molecule CCR5 coreceptor inhibitor CMPD167. 1